Method and test kit for detecting analytes in a sample

ABSTRACT

The invention relates to a method and test kit for the detection of analytes in a biological sample, wherein at least one microparticle population is labelled with fluorescent dyes serving as coding fluorescent dyes and reference fluorescent dyes and immobilized, acceptor molecules interacting with labelled analytes being bound to the microparticle population, so that the difference between the fluorescence of the microparticles and analyte(s) can be used to determine which kind of analyte, optionally in which amount, is present in the sample. In particular, the invention finds use in medical diagnostics.

The present invention relates to an efficient and inexpensive method andto a test kit for the detection of analytes in a sample.

In the meaning of the invention, analytes are understood to be chemicaland/or biological structures, said biological structures being all thosemolecules which are formed, taken up or released by organisms; chemicalstructures are understood to be all those compounds capable ofinteracting with other molecules in a way so as to allow detectionthereof. In the meaning of the invention, a sample is a materialcollected by sampling, or a portion or small amount thereof, the natureof which is to be investigated by physical, chemical and/or biologicalmeans. For example, biological samples are a portion or small quantityof serum, blood, urine, respiratory air, lacrimal fluid, or the like. Inaddition, however, samples according to the invention are subquantitiescollected from waste waters, residues from industrial processes, bogs,or from other environmental fluids.

Numerous methods of detecting analytes are known in the prior art. Inthe field of biological or clinical research and diagnostics, theanalytes to be investigated can be proteins, peptides, nucleic acids,sequence fragments, carbohydrates, lipids, and/or antigenic structures,for example.

The informative value of a parameter analysis can be expanded andimproved by parallel recording of a larger amount of data from onesingle sample—the so-called multiparameter analysis or multiligandanalysis. Parallel recording also requires miniaturization, for example,by means of which the number of detectable parameters and ligands can besubstantially increased. The miniaturized DNA technology allows foranalysis of more than 10⁶ parameters per cm², thus achieving aminiaturization degree of less than 10 μm²/parameter on one chip.Two-dimensional positioning of acceptor molecules—which moleculesinteract with the ligands—on a chip, e.g. by means of electrolithographyor other methods such as piezoelectric printing technology, makes itnecessary to repeat for each test kit the positioning procedure on thesupport material for each acceptor molecule in the same way so as toensure a regular array on the support.

However, the complex procedures required in electrolithography aresuitable only for special fields of use, e.g. in pharmacogeneticinvestigations.

As an alternative to the methods mentioned above, microparticles as DNAarray therefore have also been described in the prior art. Basically,such a microparticle array involves conjugation of multiple suspensionsof microparticle populations having different discrete fluorescencelabels with respectively specific acceptor molecules (Lackner et al.,1999, Medgen 11, pp. 16-17). Following conjugation of the acceptormolecules, the individual suspensions including the differentmicroparticle populations are mixed, and an aliquot of the mixture isadded to the sample solution so that particles of each suspension arepresent in the reaction batch in mixed state. The ligands in the samplesolution which are to be detected will bind to the correspondingacceptor molecules in a ligand-specific fashion and hence, invariably todiscrete microparticles of a particular population.

Simultaneously or subsequently, a receptor fluorescent dye is bound tothe ligands, the emission wavelength of which is sufficiently differentfrom the emission wavelength of the fluorescent dye used to label themicroparticles. The fluorescence used to identify the particles, as wellas the reporter fluorescence of ligands bound to the particles issubsequently analyzed in a flow cytometer.

Microparticles which include combinations of fluorescent dyes and can beused in various detection methods are known from the patent documentsU.S. Pat. Nos. 5,326,692 and 5,073,498. By combining fluorescent dyes,it is possible to take specific effect on the excitation and emissionwavelengths via energy transfer between different dyes incorporated bypolymerization. Furthermore, by combining different fluorescent dyes, amore specific definition of microparticle populations in a flowcytometer is possible.

However, a relatively large number of microparticles per sample, about5,000-10,000 per acceptor (Smith et al., 1998, Clin. Chem. 44,2054-2056), are required in flow-cytometric measuring methods to allowdetection of sufficient microparticles of one population in the measuredvolume. As a result, the material costs increase, which is particularlydisadvantageous in the event of expensive acceptor molecule substancesdifficult to synthesize.

Another drawback is the relatively low resolution of particlepopulations when using a flow cytometer. According to Carson et al.(1999, J. Immunol. Methods 227, 41-52), merely 64 particle populationscan be individualized when using two fluorescent dyes. Furthermore,Oliver et al. (1998, Clin. Chem. 44, 2057-2060) describe that relativelylong measuring periods of about 30 minutes and up to 1 hour per sampleare required for specific, parallel and effective detection of multipleparameters. Such long measuring periods sometimes give rise todisadvantageous modification of the ligands and fluorescent dyes.

The invention therefore is based on the object of providing an efficientmethod and a test kit allowing for shorter measuring periods and highersensitivity, which are low in cost and can be used in routine operation.

The present invention solves this technical problem by providing amethod for the detection of analytes, which method comprises thefollowing steps: fluorescent labelling of at least one microparticlepopulation, said microparticle population comprising at least onefluorescent dye serving as coding fluorescence and at least onefluorescent dye serving as reference fluorescence, binding and/orconjugating acceptor molecules to the microparticle population,immobilizing the microparticle population on a support, incubating themicroparticle population with the sample to be investigated, labellingthe analyte(s) with at least one fluorescent dye serving as reporterfluorescence, and detecting the analyte(s) by comparing the fluorescenceof the microparticle population with the reporter fluorescence.

BRIEF DESCRIPTION OF THE DRAWING

The drawing depicts a scheme illustrating the detection of antibodies ina sample.

The method of the invention comprises several steps which can bemodified in terms of their order. For example, it is possible to labelthe analytes prior to or subsequent to binding to a fluorescent dyeserving as reporter fluorescence.

If parallel investigations are to be performed with the method accordingto the invention, at least two microparticle populations are labelledwith fluorescent dyes. Microparticles in the meaning of the inventionare heterogeneous and/or homogeneous fractions of microscopic particleshaving a size of from 1 to 500 μm, particularly from 1 to 100 μm,preferably from 1 to 10 μm. The microparticles may include organicand/or inorganic components. For example, the microparticles can bepolymers which, following emulsification or boundary polymerization,precipitate on the material to be entrapped, e.g. on a fluorescent dye.The microparticles may consist of polystyrene or polyphosphoric acid,polyvinyl or polyacrylic acid copolymers. However, it is also envisagedthat the microparticles be comprised of oxidic ceramic particles such assilicon dioxide, titanium dioxide or other metal oxides. According tothe method of the invention, however, crosslinked polypeptides,proteins, nucleic acids, macromolecules, lipids, e.g. as vesicles andthe like, are also microparticles in the meaning of the invention. Thepreparation of microparticles has been disclosed in the U.S. Pat. Nos.6,022,564, 5,840,674, 5,788,991, and 5,7543,261, for example.

In the meaning of the invention, a microparticle population isunderstood to be microparticles resembling each other with respect tolabelling with the fluorescent dye or fluorescent dyes. For example, amicroparticle population may consist of microparticles labelled with adye having red, yellow or blue fluorescence and/or with fluorescent dyeswith varying lifetime of fluorescence. However, a microparticlepopulation may also be defined by the ratio of different fluorescentdyes. Thus, for example, a microparticle population may include allthose microparticles wherein the fluorescent label consists of e.g.green and red fluorescent dyes at a ratio of 1:1. The microparticles ofa microparticle population have at least one fluorescent dye serving ascoding fluorescence and at least one further fluorescent dye serving asreference fluorescence.

The coding fluorescence serves to analyze the microparticles. Themicroparticles can be labelled with different fluorescent dyes or withdifferent intensities of the fluorescent dyes. In this way, discretemicroparticle populations are formed which can be identified by means ofdetectors. In addition to the fluorescent dye(s) serving as codingfluorescence, the microparticles include at least one furtherfluorescent dye serving as reference fluorescence. The referencefluorescence allows for effective referencing of the codingfluorescence. By providing the microparticles with a referencefluorescence and a coding fluorescence, it is possible to correlate thefluorescence signals and compensate for measuring errors in this way.

Fluorescent dyes serving as label of microparticles are all thosesubstances capable of emitting detectable luminescent signals. However,it is also possible to use dyes which emit X rays or exhibitphosphorescence. Fluorescent dyes in the meaning of the invention areall those gaseous, liquid or solid inorganic and/or organic compoundswhich are characterized in that subsequent to excitation, they emit backthe absorbed energy in the form of radiation of equal, longer or shorterwavelength. That is, inorganic or organic luminescent pigments orquantum dots may also be used as fluorescent dyes in the meaning of theinvention. However, it is also envisaged that the microparticles be of anature so as to have autofluorescence or both autofluorescence and aforeign fluorescence label. For example, autofluorescence of themicroparticles can be generated by having the microparticles include themineral fluorite. For example, dansyl chloride, fluoresceinisothiocyanate, 7-chloro-4-nitrobenzoxadiazole, pyrenebutyrylaceticanhydride, N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine,1-anilinonaphthalene-8-sulfonate, 2-toluidinonaphthalene-6-sulfonate,7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole, formycin,2-aminopurineribonucleoside, ethenoadenosine, benzoadenosine, α- andβ-parinaric acid, and/or Δ^(9,11,13,15)-octadecatetraenoic acid, cadmiumselenite crystals of one single size or varying sizes and others can beused as foreign fluorescent dyes for a coding and/or referencefluorescence. As fluorescent dyes serving as reference fluorescence, itis possible to use e.g. transition metal complexes containing thefollowing substances: ruthenium(II), rhenium(I) or osmium, and iridiumas central atom and diimine ligands; phosphorescent porphyrins withplatinum, palladium, lutetium or tin as central atom; phosphorescentcomplexes of rare earths such as europium, dysprosium or terbium;phosphorescent crystals such as ruby, Cr-YAG, alexandrite, orphosphorescent mixed oxides such as magnesium fluorogermanate or cadmiumselenite crystals, fluorescein, aminofluorescein, aminomethylcoumarin,rhodamine, rhodamine 6G, rhodamine B, tetramethylrhodamine, ethidiumbromide, and/or acridine orange.

For example, the following substances can be used as fluorescent dyesfor the coding fluorescence:

-   -   ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline)/HPTS    -   ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline)/-fluorescein    -   ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline)/rhodamine B    -   ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline)/rhodamine B        octadecyl ester    -   ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline)/hexadecylacridine        orange    -   europium(III)-tris(thionyltrifluoromethyl-acetonate)/hydroxymethylcoumarin    -   platinum(II)-tetraphenylporphyrin/rhodamine B octadecyl ester    -   platinum(II)-tetraphenylporphyrin/rhodamine B    -   platinum(II)-tetraphenylporphyrin/naphthofluorescein    -   platinum(II)-tetraphenylporphyrin/sulforhodamine 101    -   platinum(II)-octaethylporphyrin/eosine    -   platinum(II)-octaethylporphyrin/thionine    -   platinum(II)-octaethylketoporphyrin/nile blue    -   Cr(III)-YAG/nile blue and    -   Cr(III)-YAG/naphthofluorescein    -   aminocoumarin/aminofluorescein    -   aminocoumarin/rhodamine 6G    -   aminocoumarin/tetramethylrhodamine    -   aminocoumarin/acridine orange    -   aminofluorescein/rhodamine 6G    -   aminofluorescein/tetramethylrhodamine    -   aminofluorescein/ethidium bromide

For example, the fluorescent dyes can be incorporated by polymerizationduring the preparation of the microparticles or subsequentlycoimmobilized on the microparticles. The fluorescent dyes can bedirectly introduced in a solvent for the microparticles, e.g. during thepreparation of the microparticles. By incorporating the fluorescent dyesby polymerization, precise determination of the amount of fluorescentdyes bound to the microparticles is possible. The fluorescent dyes canbe incorporated by polymerization in such a way that the dyes arelargely inert or undergo interaction with the analytes. Furthermore,incorporation of the fluorescent dyes in a sol-gel glass asmicroparticles with subsequent boiling, pulverizing and dispersing ofthe glass is also possible. When using pulverized fluorescent dyes, thedye can be dispersed in the form of a sensitive layer, e.g. as anexterior coating of the microparticles. For example, this can be done bycovalent or electrostatic binding of the fluorescent dyes to the surfaceof the microparticles. For example, hydroxy groups, amphiphilicelectrolytes, phospholipids, and ionic components can be used to bindfluorescent dyes to the surface of microparticles.

According to the invention, the fluorescent-labelled microparticles areintended to conjugate with or bind to acceptor molecules. According tothe method of the invention, specific acceptor molecules are coupled toeach population of microparticles. The acceptor molecules can befunctional groups such as amino groups, carboxyl groups, thiol groups,hydroxyl groups, but also, epitopes, paratopes, carbohydrates, lectins,or oligo- or polynucleotide sequences. For example, epitopes used inimmobilization can be antigenic determinants interacting with theantigen-binding portion of an antibody or with a receptor. Paratopes inthe meaning of the invention can be e.g. portions of an antibodyinteracting specifically with antigenic structures. Binding of theacceptor molecules to the respective microparticle population can becovalent, non-covalent, ionic binding or binding by other interactions.According to the invention, the microparticle populations areimmobilized on a support. As a result of such immobilization, themicroparticles are transferred into a state where reaction space islimited. In the meaning of the invention, immobilization is understoodto comprise all those methods resulting in a mobility restriction of themicroparticles by biological, chemical or physical means. The supportson which the microparticles are immobilized can be e.g. glass slides,membranes, networks, and/or fibrils. Immobilization of themicroparticles on a support may proceed either directly or via spacers.Spacers in the meaning of the invention are all those spacers capable offorming e.g. a short carbon chain between the microparticle and thesupport. For example, hydroxylated chains can be used in order to avoidspecific hydrophobic interactions. However, it is also possible toimmobilize the microparticles via the acceptor molecules. When bindingthe microparticles by means of binding sites of their own, the acceptormolecules can be selected completely at will, because they are notrequired to mediate binding to a potential support. When immobilizingthe microparticles by means of acceptor molecules, the acceptormolecules are intended to have the properties required for this purpose,such as molecular charge, chemically modifiable groups and/or immuno-,nucleic acid or hybridization affinity and the like. When immobilizingthe microparticles with the aid of acceptor molecules, immobilization bymeans of spacers is not necessarily required. Obviously, it might alsobe envisaged to immobilize the microparticles on the support via bindingsites on the surface of the microparticles.

According to the invention, at least one microparticle population isincubated with the sample to be investigated. By incubating theimmobilized microparticles and the sample to be investigated, theanalytes from the sample can interact with the acceptor molecules boundto the microparticles. If the acceptor molecules are bound antibodies,for example, the analytes—e.g. antigenic structures—can bind thereto.Because the acceptor molecules are bound to the microparticles, bindingof the analytes to the microparticles via the acceptor molecules ispossible. Advantageously, it is possible to create reaction conditionsduring incubation of the microparticle population with the sample to beinvestigated which allow for efficient interaction between the analytesand the microparticle population; such reaction conditions can beelevated temperature, for example. However, it is also possible toagitate or stir the microparticle population in the sample. Inparticular, the number of microparticle populations immobilized on thesupport is determined by the number of acceptor molecule specificitiesrequired to characterize the analytes. To this end, the desiredsuspensions e.g. are mixed, and small aliquots of the mixture arepipetted onto the support using a dispenser. The microparticles undergosedimentation inside the drop, making contact with the surface of thesupport, thereby allowing binding of 1-1.000.000, particularly1-100.000, preferably 1-10,000, and more preferably 1-1,000 or lessmicroparticles of a population to the support at random distribution.Drying of the drop on the support is to be prevented particularly inthose cases where drying of the acceptor molecules has a disadvantageouseffect on the desired binding of the analytes.

According to the invention, labelling of the analytes with at least onereporter fluorescence is envisaged. Obviously, it is possible to labelthe analytes prior to or subsequent to binding to the acceptormolecules. For example, fluorescein isothiocyanate, tetramethylrhodamineisothiocyanate, Texas red, 7-amino-4-methylcoumarin-3-acetic acid,phycoerythrin and/or cyanins, etc., or antibody-conjugated fluorescentparticles binding to the analyte e.g. by means of antibodies can be usedas fluorescent molecules. For example, direct labelling of the analyteswith a fluorescent dye is possible. By direct labelling of the analytes,it is possible to do without fluorescent-labelled antibodies or otherstructures carrying labels. In particular, direct labelling of ligandscan be performed using fluorescent dyes which emit a fluorescent signalor quench the fluorescence of other labels. However, the analytes canalso be enzyme-labelled. Examples of molecules used in enzyme labellingare horseradish peroxidase, alkaline phosphatase and/or glucose oxidase.

According to the method of the invention, detection of the analyte(s) iseffected by comparing the fluorescences of the microparticle populationwith a reporter fluorescence. In a fluorescence spectrometricdetermination, for example, the fluorescence intensities of themicroparticle population and of the analyte(s) can be compared in such away that, in particular, both the identity of the microparticlepopulation carrying analyte and the number of bound analytes can beanalyzed. Hence, it is possible e.g. to provide information as to whichanalytes are bound to particular discrete microparticle populations andin which number.

It might also be envisaged that the reference fluorescence is notinfluenced in its parameters by the analytes, while e.g. the intensityof the coding fluorescence may vary depending on the respectiveconcentration of the analytes. Thus, the signal intensity of themicroparticle population can be characterized by internal referencingwherein, in particular, an additional excitation source for fluorescenceor a second detector to determine the fluorescence is not required. Thereference quantity for internal referencing is determined on the basisof the fluorescence intensity and/or time profile of the fluorescenceresponse of the reference fluorescence. Advantageously, the codingfluorescence and reference fluorescence absorb light in the samewavelength region and therefore can be excited with the same excitationsource. In this method, the reference fluorescence is not required toexhibit a specific reaction for the molecules to be detected, butprovides a constant signal for referencing. However, it is also possiblethat both the reference fluorescence and coding fluorescence undergointeraction with the analytes. Obviously, it might also be envisagedthat neither of reference fluorescence and coding fluorescence interactwith the analytes in a way so as to modify the fluorescence signals.According to the invention, the fluorescent dyes for the codingfluorescence and reference fluorescence can be excited simultaneously bya single common source and detected together by a detector. Use of thereference fluorescence to excite the reporter fluorescence may also beenvisaged.

In one embodiment of the invention the microparticles are intended tobind to microtest plates, glass slides, flexible membranes, networks orfibrils, particularly those made of polypropylene and/or nitrocellulose,glass and/or polyvinylidene fluoride (PVDF). For example, microtiterplates are used as microtest plates. Advantageously, the microtestplates are dimensioned so as to allow use in numerous laboratoryroutines. For example, a number of fluorescence measuring instruments,such as fluorescence microscopes and the like, are designed such thatmicrotest plates can be used as standard. Thus, immobilization of themicroparticles on special laboratory vessels, e.g. microtiter plates,Petri dishes, multidishes, tray dishes, and other culture vessels andslides, advantageously allows the use of available laboratory means andapparatus for incubation, freezing, lyophilization and of similarlaboratory apparatus in clinical or research laboratories. For example,microtiter plates having a transparent, non-fluorescent flat bottompreferably can be used as microtest plates.

In another embodiment of the invention, the intention is to detect aplurality of analytes simultaneously by loading each analyte withindividual acceptor molecules via discrete microparticle populations.For example, separate loaded microparticle populations can be labelledand subsequently mixed. Aliquots of the mixture of microparticlepopulations are then contacted on a support so as to immobilize themicroparticles on the support.

Advantageously, the support is subsequently incubated with the sample sothat the analytes will bind to the acceptor molecules assigned to therespective microparticle population. Parallel detection of multipleparameters by simultaneous detection of separate analytes allows forcharacterization of a plurality of analytes with low input of materialand time.

However, binding of analytes via a linker molecule may also beenvisaged. For example, binding of the linker molecule to the analytecan be covalent or non-covalent. The linker molecule is capable ofmodulating the mobility of the analytes in such a way that the signalemitted by the analyte or by the fluorescent dye bound to the analytecan be detected efficiently.

In another embodiment of the invention, the analytes are specificallylabelled with different fluorescent dyes. The fluorescent dyes maydiffer in the color of fluorescence, fluorescence lifetime andintensities. Advantageously, a discrete microparticle population can beformed by labelling with fluorescent dyes at varying intensities, whichpopulation can be detected in a fluorescence microscope, for example.The number of possible discrete populations particularly depends on theavailable dyes and techniques of labelling the microparticles and on thenumber of colors discernable in the detection measuring instrument.Advantageously, about 60 to 100 different discrete microparticlepopulations can be produced using two colors, for example. By moreprecise determination of the intensities or by using an additionalcolor, the number of populations can be increased to about 500 to 1000well-discernable microparticle populations.

In another embodiment of the invention, the analytes are labelled withthe same fluorescent dye. Such uniform labelling of the analytesadvantageously permits determination of the total number of labelledanalytes bound to microparticles. For example, the analytes can beanalyzed by assignment to discrete populations using the labels of themicroparticles. Preferably, the fluorescent dyes and/or enzymes arepresent in monomeric and/or polymeric form. For example, the fluorescentdyes can be either inorganic compounds, such as compounds of rare earthmetals or uranium compounds, or organic compounds. Instead of labellingby means of fluorescent substrates, it is also possible to usechromogenic substrates, especially those exhibiting chemiluminescence.For sensitive detection of the analytes, binding of fluorescentmicroparticles to the analytes is also possible.

In a particularly preferred embodiment of the invention, detection ofdifferent antibodies in a serologic sample is envisaged (see FIG. 1).Microparticles exhibiting fluorescence in different colors areconjugated with respectively different antigens as acceptors andimmobilized on a support via the antigens. During the subsequentincubation, antibodies as analytes from a patient serum advantageouslybind to those antigens which are specific therefor. The bound antibodiesare detected by means of a secondary antibody having a reporterfluorescence. For assessment, the fluorescence of the microparticles andthe reporter fluorescence, especially for each image dot of a microtiterplate cavity, are measured.

The invention also comprises a test kit, said test kit comprising atleast one fluorescent-labelled, immobilized microparticle populationcapable of binding to specific acceptor molecules. According to theinvention, the immobilized microparticles comprise at least twofluorescent dyes differing in their spectral properties and/or theirlifetime of fluorescence, one dye being used for the coding fluorescenceand the other dye for the reference fluorescence. The referencefluorescence is used in referencing the coding fluorescence.Advantageously, the signals of the coding fluorescence can be correlatedto the reference fluorescence in the test kit, thereby compensating formeasuring errors. For example, the reference fluorescence allows forconfident determination as to whether one or more microparticles arelocated in the measuring area. For example, analytes to be investigatedmay also have one reporter fluorescence and one reference fluorescence.Advantageously, the ratio of coding fluorescence and reporterfluorescence can be detected more precisely by means of the referencefluorescence. In spite of the low number of microparticles used, thetest kit advantageously achieves a measuring accuracy sufficient to meetthe requirements of clinical routine, for example. Furthermore, the testkit can be designed in such a way that, owing to the immobilizedmicroparticles, the fluorescence can be evaluated using fluorescencescanners and/or fluorescence microscopes, allowing for e.g. highmeasuring accuracy and rapidity of the entire process of evaluating thefluorescence, as compared to flow cytometers, for example. The test inthe meaning of the invention can be designed in such a way that themicroparticles and acceptor molecules—solid or dissolved—are situated inseparate reaction vessels, and the fluorescent dyes and reagents forimmobilization are also kept separately. To detect an analyte, e.g.immobilization and fluorescent labelling of the microparticle populationand binding of the acceptor molecules to the microparticles are effectedin such a way that the immobilized and fluorescent-labelledmicroparticle population with the bound acceptor molecules is present inone single reaction vessel. The sample to be investigated is then placedinto this vessel. However, it is also possible to design the test insuch a fashion that all of the reagents required to detect the analytesare already present in one single reaction vessel. Advantageously, thetest of the invention permits analysis of biological and/or chemicalsamples. In biological samples such as serum, it is possible to recordmolecular parameters to characterize complex biomedical conditions, suchas immune status or genetic predisposition to specific diseases, ordetect the influence on expression. By virtue of such immobilization ofa microparticle population, it is possible e.g. to characterize a sampleincluding a small number of analytes to be investigated or ofcompetitive analytes within a very short period of time.

To determine diagnostic serologic parameters, the invention alsocomprises the use of fluorescent-labelled microparticle populationsconjugated with specific acceptor molecules, such as human or animalantibodies against infectious agents, antigens, autoantigens andallergens, pharmacologically significant binding sites in proteoms,genoms and other nucleic acids such as hormone receptors, binding sitesof pharmaceuticals, peptides, carbohydrates and DNA, to performexpression analyses of important genes and products thereof, such astumor proteins, HLA antigens, and to analyze single nucleotidepolymorphisms and mutations.

For example, one advantage of the method and test kit of the inventionis that immobilization of the microparticle populations allows to reducethe number of particles. Advantageously, this results in a moreeconomical use of the acceptor molecules compared to flow-cytometricmethods. The detection of bound analytes via e.g. fluorescentmicroparticles, quantum dots or luminescent pigments provides asensitivity down to the region of single molecules. Owing to the 3Dstructure, especially of porous particles, high and constant acceptormolecule density is achieved, which also contributes to increase thesensitivity and reproducibility. Hence, according to the invention, itis possible to reduce the number of microparticles per sample and thetime period of measuring the microparticles, thereby increasing thesensitivity of the measuring procedure. In particular, the use of areference fluorescence enables particularly easy recognition ofmeasuring areas wherein one single microparticle has been immobilized,which is especially important for automatic evaluation.

By immobilizing the microparticles on standardized supports such asmicrotiter plates or slides, it is possible e.g. to utilize availablelaboratory routines such as ELISA autoanalyzing.

Further advantageous embodiments of the invention will be apparent fromthe description.

Without intending to be limiting, the description will be illustratedwith reference to the following example.

EXAMPLE

Detection of Human IgG Antibodies Against Three MicroorganismsPathogenic in Humans

Carboxy-modified silica microparticles (sicastar®), 8 μm (micromod), orscreenBEADS, 1 μm (chemicell) having the following fluorescentproperties are prepared by adding varying amounts of fluorescent dyes tothe reaction mixture:

-   -   Microparticle population A:    -   Reference fluorescence: aminocoumarin    -   Coding fluorescence: 100% aminofluorescein    -   Microparticle population B:    -   Reference fluorescence: aminocoumarin    -   Coding fluorescence: 50% aminofluorescein    -   Microparticle population C:    -   Reference fluorescence: aminocoumarin    -   Coding fluorescence: 0% aminofluorescein

Using carbodiimide coupling, bacterial protein mixtures of Borreliaburgdorferi are coupled to the microparticle population A, of Yersiniaenterocolitican to the microparticle population B, and of Chlamydiatrachomatis to the microparticle population C. To this end,

-   1. 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is    dissolved in 1 ml of distilled water, mixed with 1 ml of bead    suspension (25 mg of beads), and incubated for 10 minutes at room    temperature;-   2. the beads are washed three times with 5 ml of MES buffer, pH 5.0;-   3. 500 μg of bacterial protein is dissolved in 1 ml of 0.1 M MES    buffer (pH 5.0) and incubated with the activated beads at room    temperature with agitation;-   4. the beads are washed three times in MES buffer, followed by    incubation in MES buffer+0.2 M glycine for 2 hours at room    temperature with agitation;-   5. the beads are washed three times in PBS, the beads are taken up    in 1 ml of PBS, aliquots of 50 μl are produced and frozen at −20° C.    for further use.

To immobilize the prepared microparticle populations on the surface of amicrotiter plate (format 384, black polystyrene, transparent flatbottom),

-   1. one aliquot of the particle suspensions of each microparticle    population at a time is thawed, mixed and diluted by pipetting 2 μl    of each suspension into distilled water so as to make a particle    density of 100 microparticles of each population per 1 μl of water;-   2. 1 μl of the mixture is pipetted into the center of the cavities    of the microtest plate;-   3. the microparticles of the three populations employed are    immobilized by drying at 45° C.

Thereafter, the cavities are washed three times with PBS+0.1% Tween 20(PBS-T). Human sera at a dilution of 1:100 in PBS-T are then pipettedinto the prepared cavities of the microtest plate and incubated for 1hour at room temperature.

Subsequently, the cavities are washed three times with PBS+0.1% Tween 20(PBS-T) and incubated for 2 hours at room temperature withgoat-antihuman IgG antiserum-phycoerythrin conjugate diluted 1:100 inPBS-T. After washing three times with PBS-T, fluorescence evaluation iseffected using an Axiophot fluorescence microscope (Zeiss). Theimmobilized microparticles are photographed successively with ablack-and-white CCD camera using optical filter pairs for the followingemission and absorption wavelengths: 390 nm/441 nm, 480 nm/520 nm and480 nm/578 nm.

Evaluation is effected by data reduction, including only those measuringareas exhibiting a reporter fluorescence intensity above a fixedthreshold value. A second data reduction/error compensation is achievedby excluding all those measuring errors from the calculation which are20% above and 50% below the reference fluorescence. The quotient of theintensities of the coding fluorescence and reference fluorescenceprovides information as to which microparticle population is concerned.Quotients deviating by more than 10% from the mean of one class resultin exclusion of that measuring area from the calculation. For eachmicroparticle population, the reporter fluorescences from 20 measuringareas are divided by the associated reference fluorescences. Theresulting quotients from these 20 measuring areas are averaged. They areproportional to the amount of human IgG specifically bound to thebacterial antigens in this microparticle population.

1. A method for determining the presence or amount of analyte(s) in asample, comprising: fluorescent labelling of at least one microparticlepopulation, said microparticle population being labelled with at leastone fluorescent dye serving as the dye producing a coding fluorescenceand at least one fluorescent dye serving as the dye producing areference fluorescence, binding and/or conjugating acceptor molecules tothe microparticle population, immobilizing the microparticle populationon a support, incubating the microparticle population with the sample,labelling the analyte(s) with at least one fluorescent dye producing areporter fluorescence, exciting the fluorescent dyes, determining thepresence or amount of analyte(s) in said sample by measuring andcomparing the coding fluorescence of the microparticles with thereference fluorescence of the microparticles to identify themicroparticle population, and measuring and comparing the reporterfluorescence of analyte(s) with the reference fluorescence of themicroparticles to correct for failures caused by varying microparticlesizes within a population.
 2. The method according to claim 1, whereinthe microparticles are immobilized on microtest plates, glass slides,flexible membranes, networks or fibrils.
 3. The method according toclaim 1, wherein a plurality of analytes are detected simultaneously byloading each microparticle population with individual acceptormolecules.
 4. The method according to claim 1, wherein several analytesare labeled with fluorescent dyes producing reporter fluorescences whichare different or identical to each other.
 5. The method according toclaim 1, wherein microtest plates, flexible membranes, networks, orfibrils which are used as support for immobilizing the microparticlepopulation, are made of at least one of polypropylene, nitrocellulose,glass and PVDF.
 6. The method according to claim 1, wherein said sampleis a clinical sample or an environmental sample.